Aberrant differentiation of progenitor cells in the hematopoietic system is known to severely impact host immune responsiveness. The infection-induced decrease in function of primary lymphoid organs has been postulated to be a mechanism promoting pathogen virulence. Our recent findings revealed that systemic infectious agents, such as T. gondii, can induce an immunocompromised state with quantitative and qualitative deficiency in naive T cells associated with long-term thymic atrophy. Moreover, toxoplasma infection triggers changes in the composition and output of bone marrow. This is exemplified in the unexpected susceptibility of NOD1 deficient mice to T. gondii challenge demonstrated previously in our laboratory. While infected NOD1-/- mice display normal serum levels of IL-12, indicative of an unimpaired innate immune response, they fail to mount an efficient IFN-gamma response. We demonstrated that this in turn was due to an impaired CD4+ T cells response resulting from an exacerbated lymphopenic state when compared to infected WT animals. Importantly we further showed that even in steady-state the thymus, spleen and lymph nodes of naive NOD1-/- mice display significantly reduced cellularity due to decreased numbers of lymphocytes (T, B and NK cells). Consistent with these findings, we found that NOD1-deficiency not only affects resistance to T. gondii infection but also anti-tumor immunity and protects mice against T cell mediated colitis. In contrast to NOD2 that is expressed primarily in myeloid cells, NOD1 is expressed ubiquitously. Bone marrow reconstitution experiments demonstrated that this deficiency stems from lack of NOD1 in the hematopoietic compartment. Subsequently, we demonstrated that intrinsic expression of NOD1 is required for optimal renewal and differentiation of hematopoietic stem cells and plays an essential role in determining the lymphopoietic potential of common lymphoid precursors. In addition to their impaired lymphopoietic potential, CD4+ T lymphocytes from NOD1-/- mice were hyporesponsive when stimulated by TCR ligand in the presence of IL-2. Interestingly, these defects were amplified in a NOD1-/- mouse strain that expresses a truncated form of NOD1 lacking the CARD signaling domain (deltaCARD NOD1) that functions as a dominant negative variant both in the presence and in the absence of the WT allele. Because STAT5 is a common and essential for signaling downstream from hematopoietic cytokines (e.g. IL-3, IL-7, CSF) as well as IL-2, we hypothesized that NOD1 expression is required for its optimal function. During the current report period we tested this hypothesis and demonstrated that NOD1 forms a complex with STAT5 in HEK293 cells transfected with both molecules and, importantly, also in primary human NK and CD4 T cells. Taken together, our findings reveal a cell intrinsic role for NOD1 in lymphocyte homeostasis and CD4+ T cell function that involves recognition of an endogenous ligand, STAT5, rather than a microbial component. More detailed analysis of STAT5 interaction with NOD1 and deltaCARD NOD1 in murine cells was hampered by lack of specific antibodies. To circumvent this problem we have now employed CRIPR technology to generate mice that express either intact or truncated NOD1 as an HA-tagged protein. As indicated from the above study, T. gondii infection creates a hematopoietic demand for lymphocytes. The expansion of hematopoietic precursors is also accompanied by an increased rate of myelopoiesis and decreased granulopoiesis. The latter effect is dependent on IFN-gamma, since when mice deficient in that cytokine are infected with T. gondii they display excessive accumulation of granulocytes at the site of infection without detectable monocyte mobilization. At present it is not clear whether IFN-gamma secreted by NK cells or Th1 lymphocytes is responsible for the suppression of granulopoiesis observed in this infection model. In FY 2019, we employed mice that cannot secrete IL-12 in response to T. gondii infection (e.g. UNC93b-/-, TLR11/12-/- and UNC93b -/-), and showed that they display decreased systemic IFN-gamma levels only at the onset of infection (3-5 days p.i. In contrast, and in agreement with our previously published data, Th1 differentiation and generation of Th1 effectors was not affected in the same mice. Nevertheless, these animals displayed profound acute mortality and accumulation of granulocytes and depletion of monocytes at the site of infection similar to that observed in T. gondii-infected IFN-gamma deficient mice. Taken together, these results strongly suggest that early NK cell derived IFN-gamma, which in contrast to Th1 cell generation is strictly IL-12 dependent, plays a unique role in promoting the hematopoietic shift towards myelopoiesis. In the absence of monocyte mobilization from bone marrow host-resistance is jeopardized despite an adequate later IFN-gamma response as these animals lack an appropriate infected cellular target to exert microbicidal activity and suppress T. gondii growth. Our previous work revealed that the mouse, although one of the natural hosts of T. gondii, may not accurately model the innate response to this pathogen in humans. In mice initiation of the innate immune response depends on expression of TLR11/12, However, humans lack functional TLR11 and the entire TLR12 gene, yet are still capable of mounting a protective immune response. As an approach for addressing this important species discrepancy we have systematically interrogated different human myeloid populations for their responsiveness to the parasite and have identified non-classical CD14+CD16+, but not classical CD14+CD16neg, monocytes as the major cells in human peripheral blood that produce IL-12 and TNF in response to T. gondii. Moreover, whereas the innate cytokine response to T. gondii in the mouse involves stimulation of TLR11/12 receptors by a soluble parasite ligand, profilin, the response of human cells requires phagocytosis of the live pathogen. In the continuation of this study we showed that the T. gondii-induced IL-12 response by CD14+CD16neg monocytes requires an additional signal that can be provided by IFN-gamma priming which is not associated with a significant metabolic shift but rather with enhanced IL-12B gene transcription. Interestingly, despite comparable expression of IFN-gamma receptor, IFN-gamma failed to enhance the cytokine response of the CD14+CD16pos subset. In work performed during the current report period, we showed that the IFN-gamma hypo-responsiveness of classical monocytes is associated with an IFN-induced gene transcriptional profile that is displayed ex-vivo at base line before stimulation. A similar dichotomy between monocyte subsets was observed when rapamycin, an inhibitor of the mTORC pathway was used as secondary signal to prime the IL-12 response of the CD14+CD16neg population. Taken together, our results demonstrate that while CD14+CD16+ non-classical monocytes are poised to produce IL-12 in response to microbial stimuli, classical monocytes require an additional priming signal that can be provided by IFN-gamma or rapamycin.